1. Field of the Invention
The present invention relates to methods of using the chromium(III) complex represented by the formula [Cr.sub.3 O(O.sub.2 CCH.sub.2 CH.sub.3).sub.6 (H.sub.2 O).sub.3 ].sup.+ to reduce plasma levels of cholesterol and triglycerides. The invention also includes compositions which contain this chromium(III) complex.
2. Background of the Invention
In the late 1950s and 1960s, rats fed a chromium-deficient diet were found to possess a decreased ability to repress blood glucose concentrations, while chromic ions were shown to increase the efficiency of insulin action in rat epididymal tissue [1-5]. Since these observations, a search has been underway to identify the biologically active form of chromium, that is, the biomolecule which naturally binds chromium (III) and possesses an intrinsic function associated with insulin action in mammals [6-8]. Subsequent demonstration that the populations of developed nations intake on average less than the recommended safe and adequate amount of chromium in their daily diet [9, 10] has resulted in the development of chromium-containing dietary supplements. Such materials also have potential as insulin-potentiating therapeutics which could possibly see use in the treatment of diabetes [11]. Determining the structure, function, and mode of action of the biologically active form of chromium could greatly aid in the rational design of such potential therapeutics.
The first chromium-containing species proposed to be biologically active was glucose tolerance factor(GTF)[1,12]. GTF was first isolated from acid-hydrolyzed porcine kidney powder, although a similar, if not identical, material was subsequently isolated from yeast[1,13]. Currently the term GTF is usually understood to refer to only the material isolated from yeast. GTF is absorbed better than simple chromic salts and potentiates insulin action in rat epididymal tissue or isolated rat adipocytes [14]. However, kinetics studies indicate that GTF does not intrinsically possess biological activity [15]; additionally, the material is apparently a byproduct of the acid hydrolysis step used in its purification [16].
GTF was proposed to be composed of chromic ion, nicotinic acid, and the amino acids glycine, glutamic acid and cysteine [13]. While these results have not been reproducible in some laboratories [17-21], this report stimulated an intense interest in the synthesis of chromic-nicotinate complexes [22-25], some of which have been patented as nutritional supplements. The proposed identification of nicotinic acid (2-carboxypyridine) also stimulated investigations of complexes of chromium(III) with the related pyridine carboxylic acids picolinic acid (2-carboxypyridine) and isonicotinic acid (4-carboxypyridine) [26-28]. As a result chromium(III) tris(picolinate), Cr(pic).sub.3, has become a very popular nutritional supplement and is being tested as a therapeutic for the treatment of symptoms of adult-onset diabetes. It is available over-the-counter in the form of pills, chewing gums, sport drinks, and nutrition bars. Cr(pic).sub.3 is also a well absorbed form of chromium and has been proposed to be the biologically active form of chromium [29]. This is, however, extremely doubtful given the chemistry required to synthesize this material.
In the last decade, a number of investigators have examined the effects of administering Cr(pic).sub.3 (and in some cases other forms of chromium(III)) to rats on regular diets [30-33]. After an initial preliminary report which suggested beneficial effects on blood variables [30], detailed examinations of the effect of Cr(pic).sub.3 administration in amounts up to 1500 .mu.g/kg diet for up to 24 weeks have found no acute toxic effects [31-33]. However, the compound and other chromium sources examined (most notably "Cr nicotinate" and chromium chloride) also had no effect on body mass, percentage lean or fat content, tissue size (heart, testes, liver, kidney, muscle, epididymal fat, spleen, and kidney), or blood variables (fasting glucose, insulin, cholesterol, etc.). No differences in the gross histology of the liver or kidney (organs where chromium(III) preferentially accumulated) were found, although chromium did accumulate in these organs [33]. Another study compared the effects of a Cr-deficient diet with diets supplemented with ten different sources of chromium, including allowing rats to live in stainless steel cages. The Cr sources had no effect on body mass; all but one source decreased epididymal fat. Testes and liver masses tended to be lowered, whereas kidney, heart, and spleen masses were not significantly altered. Supplemental Cr had no effect on serum triglycerides or cholesterol, and only one source resulted in lower serum glucose [34]. While these studies did not manifest any acute toxicity, the lack of beneficial effects of Cr(pic).sub.3 supplementation on growth, fat content or glucose, insulin, or cholesterol concentrations raises questions about its therapeutic potential. Recently the safety of intaking Cr(pic).sub.3 has been questioned, especially in regards to its potential to cause clastogenic damage [35,36]. At physiologically-relevant concentrations of chromium (120 nM) and biological reductants such as ascorbic acid and thiols (5 mM), Cr(pic).sub.3 has been shown to catalytically produce hydroxyl radicals which cleave DNA[35]. This ability stems from the combination of chromium and picolinate; the picolinate ligands prime the redox potential of the chromic center such that it is susceptible to reduction. The reduced chromium species interacts with dioxygen to produce reduced oxygen species including hydroxyl radical. These studies are in agreement with earlier studies which showed that mutagenic forms of chromium(III) required chelating ligands containing pyridine-type nitrogens coordinated to the metal [37].
Recently the naturally-occurring oligopeptide low-molecular-weight chromium-binding substance, LMWCr, has been proposed as a candidate for the biologically active form of chromium [6,7,38,39]. Kinetics studies of insulin action on rat adipocytes suggest that LMWCr has an intrinsic function in insulin-sensitive cells [15,40]. The oligopeptide appears to be part of an insulin signal amplification mechanism [6,7]. The oligopeptide containing four chromic ions binds to insulin-activated insulin receptor, stimulating its tyrosine kinase activity up to eight-fold with a dissociation constant of approximately 100 pM [38]. Spectroscopic studies have shown that LMWCr possesses a multinuclear chromic assembly where the chromic centers are bridged by anionic ligands (presumably oxide and/or hydroxide). The assembly is supported by carboxylate groups from aspartate and glutamate residues from the oligopeptide [41]. This discovery has spurred an interest in the synthesis and characterization of multinuclear oxo(hydroxo)-bridged chromium(III) carboxylate assembles [42-45]. In 1997, such an assembly, [[Cr.sub.3 O(O.sub.2 CCH.sub.2 CH.sub.3).sub.6 (H.sub.2 O).sub.3 ].sup.+, 1, was found to mimic the ability of LMWCr to stimulate insulin receptor kinase activity [39]. Both LMWCr and the biomimetic 1 have been proposed as potential nutritional supplements and therapeutics. Both LMWCr and 1 have been shown not to lead to DNA cleavage [46]. The synthetic complex has several potential benefits over the natural material: it is inexpensive to synthesize and can be readily prepared in bulk. LMWCr is susceptible to hydrolysis, especially in the presence of acid, whereas the synthetic material can be recrystallized from dilute mineral acid [47] and could potentially survive oral ingestion. After the insulin signaling event, LMWCr may be excreted in the urine [48,49], and it is possible the body might target the material for excretion rather than absorption.